The role of four aromatic residues (W85, Y172, W266 and W274) in the struct
ure-function relationship in xylanase A from Streptomyces lividans (XlnA) w
as investigated by site-directed mutagenesis where each residue was subject
ed to three substitutions (W85A/H/F; W266A/H/F; W274A/ H/F and Y172A/F/S).
These four amino acids are highly conserved among family 10 xylanases and s
tructural data have implicated them in substrate binding at the active site
, Far-UV circular dichroism spectroscopy was used to show that the overall
structure of XlnA was not affected by any of these mutations. High-performa
nce liquid chromatographic analysis of the hydrolysis products of birchwood
xylan and xylopentaose showed that mutation of these aromatic residues did
not alter the enzyme's mode of action. As expected, though, it did reduce
the affinity of XlnA for birchwood xylan. A comparison of the kinetic param
eters of different mutants at the same position demonstrated the importance
of the aromatic nature of W85, Y172 and W274 in substrate binding. Replace
ment of these residues by a phenylalanine resulted in mutant proteins with
a K-M closer to that of the wild-type protein in comparison with the other
mutations analyzed. The kinetic analysis of the mutant proteins at position
W266 indicated that this amino acid is important for both substrate bindin
g and efficient catalysis by XlnA. These studies also demonstrated the cruc
ial role of these active site aromatic residues for the thermal stability o
f XlnA.